Cryptorchidism is by far the most common defect of sexual differentiation in humans. Testicular descent can be anatomically divided into two stages, i.e., transabdominal and inguinoscrotal phases. It is known that both insulin-like peptide 3 (INSL3) and androgen signaling pathways play critical roles in the control of the transabdominal phase of testis descent. However, the mechanism that regulates the inguinoscrotal phase is not completely understood, even though the incidence of this defect is much more common clinically. Targeted disruption of luteinizing hormone receptor (LHRKO) in mouse impaired development of adult-type Leydig cells which resulted in a dramatic reduction of INSL3 and testosterone levels. Surprisingly, serum estradiol levels were significantly increased. LHRKO males exhibited a bilateral cryptorchid phenotype resulting from a defect in inguinoscrotal testis descent. Histology, morphometry, cell proliferation and differentiation analyses demonstrated the defect was due to a reduction in mesenchymal cell division and differentiation into cremaster muscle cells during the second stage of testis descent. The expression of several genes in the gubernaculum that are known to be involved in gubernacular development, such as Hoxa10, Hoxa11, Wt1, Dll1 and Desrt, were not altered in mutant mice, while Lgr8, Notch1 and Numb mRNA levels were drastically deceased as compared with age-matched wild type siblings. In contrast, the expressions of Esr1 (ER1) and Esr2 (ER2) were significantly elevated. Postnatal testosterone replacement therapy (TRT) completed testicular descent into the scrotum and concomitantly normalized estradiol concentrations in the circulation and Esr1, Esr2, Lgr8, Notch1 and Numb mRNA levels in the gubernaculum. Remedy of the defect by TRT was neither dependent on recovery of adult-type Leydig cells nor the genitofemoral nerve but required androgen receptor activity in the gubernaculum. Using organ culture of the gubernaculum in vitro, androgen alone did not significantly stimulate proliferation of gubernacular mesenchymal cells without activation of the leucine-rich repeat-containing G-protein coupled receptor 8 (LGR8) signaling pathway by addition of INSL3 or relaxin. Co-treatment of estrogen with androgen prevented androgen-induced Lgr8 expression. Cotreatment of INSL3 and androgen increased Notch1 but not Numb mRNA levels. Taking these results together, we hypothesize that induction of the Lgr8 gene expression by androgen and activation of LGR8 signal impinging on the Notch system to stimulate gubernacular cell proliferation, differentiation and myogenesis are the essential molecular pathways in the control of gubernacular development to facilitate inguinoscrotal testis descent. In addition to androgen deficiency, unbalanced androgen/estrogen signals also play a role in contributing to the cryptorchid phenotype in LHR null animals. In this grant application, we propose four specific aims to verify our hypotheses: (1) to define the underlying mechanism by which androgen modulates the Lgr8 gene expression in the gubernaculum; (2) to assess the role of the LGR8 signaling pathway in androgen-induced inguinoscrotal testis descent in LHR null males; (3) to determine the importance of the Notch signaling pathway in mediating androgen and LGR8 signals to regulate gubernaculum development during inguinoscrotal testis descent and (4) to investigate the effect of estrogen on androgen-induced inguinoscrotal testis descent in LHR null males. Cryptorchidism is by far the most common birth defect in males. Although inguinoscrotal testis descent is known to be androgen dependent, the underlying mechanism remains unclear. The current proposal is focusing on determining androgen downstream target genes and subsequent signaling network during the process of inguinoscrotal testis descent, which may provide new insight into the causes of testis maldescent.